National Center for Design of
Biomimetic Nanoconductors

• Research:
• Overview

Research Overview

Introduction

The overall goal of the NIH Nanomedicine Centers program, as stated on the program Web site, is to use the knowledge that researchers have already developed to “extensively categorize the parts of cells in vivid detail to answer questions such as, "How many?" "How big?" and "How fast?"  These questions must be addressed in order to build "nano" structures or "nano" machines that are compatible with living tissues and can safely operate inside the body. Once these questions are answered, we will design better diagnostic tools and engineer structures for more specific treatments of disease and repair of tissues.”

The parts of the cell with which our National Center for Design of Biomimetic Nanoconductors is specifically concerned are native and mutant biological channels and other ion transport proteins and synthetic channels, and heterogeneous membranes containing channels and transporters. The model experimental systems are engineered protein channels and synthetic channels in isolation, and in self-assembled membranes supported on nanoporous surfaces.  There are many scientists in the world who study biological membranes, including those who use that knowledge to design therapies.  However we fill a unique niche, in that our team studies membranes as systems to be engineered.

Conceptual Background

The creation of structures at the nanoscale can in general be done by two classes of methods, top-down nanofabrication, (shrinking larger scale processes down to the nanoscale, such as in nanolithography) and bottom-up nanofabrication, (inducing spontaneous formation of desired structures).Top-down nanofabrication technology is at this writing relatively well-developed because it is a direct extension of previously established technologies.  However bottom-up nanofabrication technology is believed to have enormous future utility because of the potential for very high degrees of molecular-level specificity of interactions that can be enabled with this approach, and also because of the generally smaller dimensions that can be readily achieved. 

The aspects of our Center that are novel mainly employ bottom-up approaches.   A key concept underlying bottom-up approaches in general, and our Center in particular, is self assembly, (the ordering of molecules into well defined structures by noncovalent bonding). The proof of concept that useful structures can be created at the nanoscale by the mechanism of self assembly comes from biology.  For example proteins, which are a few nanometers in size, acquire their 3-dimensional structures by a process called protein folding, a self assembly process.  As another important example, the surfaces of biological cells and organelles (functional compartments within cells) are formed by lipid bilayer membranes, which are also formed by self-assembly.  These membranes are approximately ten nanometers thick.   Some proteins reside in the cell interior and others reside in cell and organelle membranes. The insertion of proteins into membranes is also a self assembly process.  The noncovalent forces governing self assembly of biomolecular structures are of two sorts: 1) Coulomb forces, which are simply the mutual attraction and repulsion of unlike and like charges respectively, and 2) the van der Waals force, which has two components.  The short range component of the van der Waals force is a hard sphere repulsion, which stems from the fact that the electron clouds of atoms that are not covalently bonded cannot overlap.  The long range component of the van der Waals force is attractive, and is physically caused by the dipole moments that atoms induce in each other.  Noncovalent forces in the context of an aqueous medium give rise to the hydrophobic effect, which means that in aqueous environment polar molecules or regions of molecules tend to become exposed to the water while non-polar molecules or regions tend to pack with each other.  The hydrophobic effect results in the phenomenon that the interiors of proteins and membranes are less polar than the regions on the surface.

Some proteins require the assistance of other proteins (molecular chaperones) to fold correctly.  Spontaneous self-assembly of a particular structure does not always produce the desired result, either in natural systems, experimental systems, or in a potential nanoscale device.   The key to utility at the nanoscale is directed self assembly, by which we mean self assembly under conditions that will produce a useful and desired structure.

Assembling the NDC team

Realizing the potential of supported membranes in particular, and directed self-assembly in general, is far beyond the scope of any individual laboratory and any particular scientific or technical discipline.  Because the most powerful and compelling examples of useful self-assembled structures are biological, the project needs to include a strong molecular biophysics component.  Manipulation of materials at the nanoscale requires a strong nanotechnology component.  Engineering proteins requires a strong biochemistry and synthetic chemistry component.  Understanding the structure of materials requires a strong materials science component.  Because the theoretical and computational aspects of self assembled systems are still in a developmental stage, a strong theory and computation component is required for the team, focusing on the areas of transport through channels and membrane organization.

NDC Aims

Membrane transport accomplishes several basic functions in biological systems including: a) Electrical and electrochemical signaling, b) generation of osmotic pressures and flows, c) generation of electrical power, and d) energy transduction, and e) molecular recognition and chemical signal transduction.  Our specific aims are two fold.  We wish to

1. Engineer nanoscale systems with synthetic membranes that accomplish the functions of natural membranes in the service of therapy.
2. Combine a systems understanding of membranes with an ability to manipulate the molecular components of membranes to repair defective natural membranes.

Progress Highlights

We have invented and created the first prototype of the Functional Protocell

We define the “functional protocell" as a nanoporous solid surrounded by a membrane. The cavities in the solid can be filled with any desired electrolyte up to the limit of solubility. The surrounding membrane can contain any combination of membrane proteins. Thus the functional protocell can be imbued with any array of intracellular and membrane processes that are desired. It can be considered analogous to either a biological cell or an organelle such as a mitochondrion or a chloroplast (which started life as bacteria the order of 1018 nanoyears ago).

The phrase “functional protocell” as we are using it should be distinguished from the term “minimal protocell” that is commonly applied to a (so far hypothetical) minimal assembly of molecules that would have all the essential properties of cellular life, including self-replication. The “functional protocell” does not have all the essential properties of life, but would have specific designed properties that would make it technologically or biomedically useful. In some ways, the functional protocells would have the basic components of cells---a membrane that could do molecular recognition and transport, and an intracellular network of reactions that could sustain cell-like functioning. But it would have no properties that are not specifically built into it; especially the “functional protocell” would not self-replicate.  The rationale for creating the functional protocell stems from the observation that cells have enormous flexibility and specificity of behavior compared to human-made entities on the same size scale. The key is the combination of a complex surface coating for specific molecular recognition and transport tasks, and complex contents comprised of networks of molecules organized to do specific tasks. Biological cells and viruses are often questionable therapeutic agents because of unpredictable side effects. Living cells and viruses may be modified by engineering to give some desirable properties, but because their functioning and their effects on human function are not completely understood, undesirable side effects can not be fully predicted and eliminated. Completely synthetic functional protocells, on the other hand, would only have components that are put into them, and could be guaranteed not to replicate.

Prior to the initiation of the NDC, the laboratory of Jeff Brinker (one of our NDC investigators) had developed a technique for creation of nanoporous silica objects, either as films or discreet spheres, with a size ranging from a few hundred nanometers to a micron (roughly the span from a virus to a small cell. The laboratory then acquired the ability to load the nanoporous spheres with any desired set of reactants in aqueous medium, by immersing the nanoporous spheres in a solution of the reactants, and then causing the reactants to be concentrated in the interior of the nanospheres by drying, as shown below:

Within the past year, the Brinker lab succeeded in coating the nanoporous sphere in a protein-containing membrane by deposition of liposomes on the surface of the nanoporous sphere as shown below:

The applications we envisage for the functional protocell will be given in a later section.

We have developed a computational method for simulating and predicting domain formation in heterogeneous membranes.

All biological membranes are made up of multiple types of molecules.  Membranes made of more than one type of molecule will tend to form membrane domains, meaning that different parts of the membrane will have somewhat different properties.    Membrane domain formation has high biological significance.  The functioning of membrane proteins is modified by protein-lipid interactions thatdepend on the local lipid environment around the protein.  Up to now, it has not been possible to rigorously simulate domain formation, because of how much computing time is necessary for atomically detailed molecular dynamics simulations of such a large system.  Larry Scott of the NDC has developed a novel statistical mechanical method in which the results from atomically detailed molecular dynamics simulations of membranes are used to compute a mean interaction field for the different membrane components.  The mean interaction field is utilized in a Langevin dynamics program.  Scott and his co-workers have shown that for a phospholipid-cholesterol mixture (one of the most important classes of mixtures in membranes) this method successfully replicates the thermodynamic and structural properties of the mixed membranes.  This prototypical result opens the way to predict and describe on an atomic level of detail domain formation in synthetic and natural membrane systems.

We have developed a covalently linked alpha-hemolysin and cyclodextrin anion-selective channel.

Alpha-hemolysin is a protein ion channel with a very large pore that is secreted by a bacterium and functions as a toxin, by inserting itself into cell membranes and permitting indiscriminate passage water and ions, essentially short-circuiting the cell membrane’s role in regulating traffic between the cell interior and the exterior.  Cyclodextrins are cyclic sugar molecules; informally one might describe them as shaped somewhat like donuts.  Previously the Hagan Bayley lab of our NDC had shown that a cyclodextrin molecule would spontaneously insert into the lumen of an alpha-hemolysin channel and, while there, cause a reduced conductance moderately selective to anions.  Now the Bayley lab has acquired the ability to covalently link the cyclodextrin into the channel lumen, creating a permanent anion-selective structure.  This provides a platform for engineering channels of custom selectivity, by attaching various groups to the cyclodextrin.  The first target will be a strongly chloride-selective channel, by addition of amine groups.

Other Advances

• We constructed a quantitative model of the eel electric organ that can be used as an engineering design tool for a nano-engineered biocompatible battery. (LaVan lab)

•  We succeeded in generating an electrical potential across a network of linked synthetic cells, an important proof-of-concept milestone towards the nano-engineered biocompatible battery (Bayley lab)

• We created a theoretical framework for understanding (and hopefully predicting) specific ion selectivities, such as the selectivity between sodium and potassium (Roux lab and Rempe lab)

• We succeeded in creating an addressable array of membrane patches on nanoporous supports to serve as a foundation for high speed assays of membrane function and sensing devices involving membrane molecules (collaboration between the Parikh and the Brinker labs).

Applications

“Smart” hemodialysis utilizing functional protocells:

 In this application blood would be passed through a filter containing functional protocells. The protocell membranes would be equipped with proteins that would:

• use active transport to accelerate the passage of materials that pass through existing dialysis membranes, such as urea and potassium ions.

• take up toxic materials that may be present in the blood in low but damaging concentrations. A notable possibility here is to deploy in the smart membrane detoxification proteins from bacteria or from plants to cleanse the blood of toxic metals. Essentially this would extend the technique of phytoremediation (using biological transport to clean up pollution) to dialysis.  Here the application is not limited to renal failure (the condition for which hemodialysis is most often used), but could be used to remediate other poisoning situations such as lead.  After a dialysis session the protocells would be either “recharged” (if feasible), or replaced

Combating Infection utilizing functional protocells

In this mode of functioning the protocell would have a surface coating that would recognize and bind to viruses or other pathogens. Once bound, a reaction would be triggered that would neutralize or kill the pathogen.  One can reasonably envisage several modes of functioning, as follows:

• The protocells would be deployed in the airway or the GI tract, for pathogens that reside in those spaces.
• The protocells could be injected and exert their action in extracellular fluid.
• The protocells could be injected and engineered to be taken into the interior of cells, in the manner of a virus.  These “benign invaders” would target pathogens inside cells and could deliver drugs to cell interiors.
• The protocells could be used in a hemodialysis machine. In this instance the blood would be passed through a filter containing a suspension of honeypot protocells. Blood borne pathogens would be removed by association with the surface membranes of the protocells. An obvious disadvantage of the dialysis mode would be the need to connect the patient to the machine for the treatment. On the other hand, a great advantage to the dialysis mode would be that the protocells themselves would not enter the general circulation, so that there would not be concerns about the side effects of residual protocell contents in the body.

Building a Biocompatible Battery that would be recharged by biological metabolism

This was our founding mission, and we have made progress in the last year on both experimental and theoretical fronts. On the experimental front we have been able to create a network of synthetic cells that produces a voltage from one side of the network to the other. On the theoretical side we have made a dynamical model of the electric organ of the electric eel (the biological proof of concept for the biobattery) that can serve as an engineering design tool for the synthetic biobattery.  However the functional protocells of the biobattery, and the scaffold to house them, constitute exceptionally difficult problems relative to other functional protocell applications, because it requires the development of polarized cells (cells that have two distinctly different sides) that are in a specified geometric relationship to each other.

Direct protein therapy for cystic fibrosis

The common genetic defect in cystic fibrosis is in a gene that codes for the chloride-selective channel CFTR. While the gene is expressed in several epithelia (tissues composed of a layer of cells), the lethal symptoms are in the airway. The airway mucus becomes drier and more viscous, and thus cannot be moved by the cilia in the airway towards the mouth to clear inhaled foreign materials. The mucus becomes susceptible to opportunistic infection. Researchers in NCDBN have acquired the ability to convert a bacterial toxin, alpha-hemolysin, into an anion selective ion channel. The cystic fibrosis initiative within NCDBN is to explore the question of whether this finding by NCDBN can be the basis of direct protein therapy, in which a designed synthetic protein channel would play the role that the defective CFTR can not.

Understanding oxidative damage to membranes

In another medical-directed initiative, NCDBN is utilizing an experimental technique developed by us (individually addressable arrays of membrane patches on a single nanoporous surface) and a computational technique newly applied by us to membranes (Mean Field Langevin Dynamics) to study in more detail than has previously been possible the changes in biological membrane organization that accompany oxidative stress. Changes in membrane properties under oxidative stress are implicated in many diseases, so we believe that the basic knowledge that we generate will ultimately be medically applied.  Our initial focus is on oxidative membrane damage in macular degeneration and atherosclerosis.

References

Background (More background references available in the bibliography):

1. Eric Gouaux and Roderick MacKinnon 2005 Principles of Selective Ion Transport in Channels and Pumps Science 310: 1461-1465
2. Sushmita Mukherjee, Frederick R. Maxfield.  2004 Membrane domains Annual Review of Cell and Developmental Biology 20, 839-866
3. F. Ratjen, G. Döring.  2003 Cystic fibrosis.  The Lancet, Volume 361, Issue 9358, Pages 681-689
4. Motomu Tanaka, Erich Sackmann 2006 Supported membranes as biofunctional interfaces and smart biosensor platforms Physica Status Solidi (a) V 203, N 14, 3452-3462

Current NDC Supported Papers (more supported papers available in publications):

1. S. Varma & S. B. Rempe, 2007 Tuning ion coordination architectures to enable selective ion permeation. Biophys J. 93:1093-1099.

2. Hwang WL, Holden MA, White S, Bayley H 2007 Electrical Behavior of Droplet Interface Bilayer Networks: Experimental Analysis and Modeling J Am Chem Soc.

3. Pandit SA, Khelashvili G, Jakobsson E, Grama A, Scott HL. 2007 Lateral organization in lipid-cholesterol mixed bilayers. Biophys J. 2007 92:440-7.

4. Baca, Helen; Carnes, Eric; Singh, Seema; Ashley, Carlee; Lopez, DeAnna: Brinker, C. Jeffrey, 2007 Cell-directed assembly of bio/nano interfaces – a new scheme for cell immobilization  Accounts of Chemical Research

 

The National Center for Design of Biomimetic Nanoconductors is funded by Grant Number PHS 2 PN2 EY016570B from the National Institutes of Health through the NIH Roadmap for Medical Research and is based at the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. Partner institutions include Yale University, University of Southern California, University of Chicago, Illinois Institute of Technology, Sandia National Laboratories, Argonne National Laboratory, Oxford University, Wabash College, University of California, Davis, nanoHUB at Purdue University, and the University of New Mexico.